We are developing a gene targeting program based on the capacity of oligonucleotides to form stable triple helix complexes with specific sequence in duplex DNA. This approach has the promise to become a simple and efficient technology for delivering DNA reactive compounds to specific sites in chromosomal DNA in living cells. Applications include gene knockout, directed gene conversion and recombination, and, perhaps, gene therapy. Triple helices have been known for over 40 years and have been the subject of many studies in vitro. However there is direct evidence that the protein:nucleic acid structure of mammalian chromosomes would preclude access to triplex forming oligos (TFO). We prepared a TFO linked to a photoactivatable DNA mutagen directed against a sequence in a gene (HPRT) frequently used as a mutation reporter. We introduced this into mammalian cells and after photoactivation of the mutagen isolated colonies of cells with mutations in the target gene. Sequence analysis showed that the mutations were located at the target sequence within the gene. We have prepared TFOs with novel sugar modifications that show enhanced targeting activity. Treatment of cells with these TFOs yields clones with targeted mutations at frequencies around 5%. These results indicate that chromosomal target sites in mammalian cells are accessible to these reagents, and open the door to wider application of these novel reagents.